Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.
The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMé rieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.
Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.
Most RNA viruses have evolved mechanisms to avoid neutralizing antibody responses, and it is generally believed that variability of envelope-encoding regions is the major molecular basis of this phenomenon. However, it has been hypothesized that other mechanisms can be involved. Recent experimental data indicate that in hepatitis C virus (HCV) infection, the anti-envelope humoral response includes cross-reactive antibody clones able to neutralize vesicular stomatitis virus (VSV) pseudotypes containing HCV E1 and E2 glycoproteins (HCV/VSV pseudotype) as well as other clones devoid of such activity. In this work, we demonstrate that natural infection with a large variety of HCV isolates belonging to different genotypes elicits HCV/VSV pseudotype-neutralizing cross-reactive anti-envelope antibodies together with clones unable to neutralize this pseudovirus. This was shown by designing a novel strategy for quantitation of serum antibodies binding selectively to single viral cross-reactive conformational epitopes. These data can be useful not only for a better understanding of the virus-host interplay in important viral diseases, but also for the development of an effective anti-HCV vaccine.
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